PL248990B1 - Method of determining the risk of death in women with breast cancer depending on serum zinc concentration - Google Patents

Abstract

The subject of the application is a method for determining the risk of death in women with breast cancer, characterized in that it includes a quantitative assessment of the concentration of zinc in the serum of the examined person, where the concentration indicates a 3-fold reduced risk of death in relation to the subgroup with the lowest concentration of zinc in the serum, in the case of serum zinc concentration values above 935 μg/l.

Description

Description of the invention

The invention relates to a method for determining survival in breast cancer patients based on serum zinc concentration. The described invention is based on the finding that there is a correlation between serum zinc concentration and survival in breast cancer patients. The presented method should be used to support and complement traditional treatment for breast cancer patients.

Zinc is a trace element essential for health. It is a component of approximately 300 enzymes and an even larger number of other proteins [1]. As a component of so-called zinc fingers, protein domains found in DNA-binding proteins, it influences cellular life processes. Zinc protects against free radicals, including as a component of superoxide dismutase (SOD). It participates in numerous metabolic processes, being a component of enzymes such as malate dehydrogenase, carbonic anhydrase, lactate dehydrogenase, and alcohol dehydrogenase. This trace element is involved in immunological processes, determines the proper function of skin and mucous membranes, participates in the storage and secretion of insulin from the pancreas, maintains the ionic balance of other trace elements, including selenium, magnesium, and copper, and also detoxifies heavy metals. Zinc is an antioxidant, contributing to defense against oxidative stress. Zinc is considered a non-toxic metal. Deficiency of this element leads to serious disorders such as immune deficiencies, impaired wound healing, decreased fertility, and vision problems [2].

Zinc levels have been observed to be altered in cancer cells. Normal prostate epithelial cells accumulate zinc, whereas cancer cells have significantly reduced levels of this element [3]. Zinc is believed to have anticancer effects by inhibiting cancer cell growth and activating apoptosis. Studies examining the relationship between zinc levels and cancer risk have been conducted, but their results are inconsistent. Some studies indicate that serum zinc levels are higher in individuals with cancer [4,5,6], while others indicate lower levels [7]. Previous studies also suggest that adequate dietary zinc has a chemopreventive effect. Individuals whose diets are rich in zinc have a lower risk of lung cancer than those whose diets are low in zinc (OR 0.71; 95% CI 0.5-0.99) [8]. The risk of colon and rectal cancer is also lower with a zinc-rich diet (RR 0.86; 95% CI 0.73-1.02) [9]. However, zinc supplementation at very high doses above 100 mg/day (when the recommended daily intake of zinc is 8 mg/day for women and 12 mg/day for men) has the opposite effect, significantly increasing the risk of prostate cancer (RR 2.29; 95% CI 1.06-4.95, p=0.03) [10].

The above data address the importance of zinc as a cancer risk factor. There is considerable evidence suggesting a correlation between the functioning of zinc-dependent proteins and the survival of cancer patients. However, none of these studies demonstrate the importance of serum zinc concentrations in predicting cancer patient survival [11-15]. Therefore, this study aimed to assess the correlation between the risk of death in women with breast cancer and serum zinc concentrations. Unexpectedly, such a correlation was found.

The subject of the invention is a method for determining the survival of patients with breast cancer, characterized in that it comprises a quantitative assessment of the concentration of zinc in a serum sample of the examined person, wherein the concentration of zinc > 935 pg/l indicates a more than several-fold reduction/increase in the risk of death of patients with breast cancer when assessing 10-year survival.

Preferably, the sample material is serum.

Preferably, the zinc concentration in the sample is determined by direct measurement in serum.

An example of an invention implementation

Study group

The study included 283 patients from Independent Public Clinical Hospitals No. 1/2 and 255 patients from the West Pomeranian Oncology Center with diagnosed and histopathologically confirmed breast cancer. Informed consent was obtained from each patient, a blood sample was collected, and pedigree and clinical data were obtained. Blood was collected between 2009 and 2015 at the time of breast cancer diagnosis but before treatment. Patients were instructed to fast for at least 4 hours before collection. In 2020, information on deaths during the prospective follow-up was collected from the Department of State Records of the Ministry of Digital Affairs. The average follow-up period for surviving patients was 9 years (range 5-12 years).

PL 248990 Β1

The characteristics of the group are presented in Table 1.

Table 1. Group characteristics

Living Deaths Average age (range) 55.87 (25-85) 61.07 (28-91) Smoking cigarettes -Currently 89 (21.3%) 26 (21.5%) -in the past 112 (26.9%) 27 (22.3%) -Never 206 (49.4%) 65 (30.7%) -no data 10 (2.4%) 3 (1.4%) Estrogen receptor -positive 287 (68.8%) 79 (65.3%) -negative 114 (27.3%) 32 (26.4%) -no data 16(3.8%) 10(8.26%) Treatment -chemotherapy 211 (50.6%) 65 (53.7%) -radiotherapy 244 (58.5%) 56 (46.3%) -tamoxifen 282 (67.6%) 79 (65.3%) Mutation in the BRCA1 gene 50(12%) 11 (9.1%) Type of surgery -mastectomy 261 (62.6%) 82 (67.8%) -lumpectomy 131 (31.4%) 17 (14%) -no data 25 (6%) 22 (18.2%)

Material

A blood sample was collected from each person included in the study to measure zinc concentration. After collection, the sample was stored at -80°C until zinc concentration was determined.

Method for determining zinc content in serum

1.1 Camera

Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine zinc content. The measurements were performed using an ELAN DRC-e mass spectrometer (PerkinElmer) and a NexlON 350D (PerkinElmer). ICP-MS allows for detection limits of <0.1 pg/L. Instrument sensitivity is crucial when conducting assays in populations not occupationally exposed to metals and their compounds.

1.2 Preparation for measurement

Collected serum samples were thawed from -80°C to room temperature on the day of analysis. Each sample was thoroughly mixed using a shaker or vortex mixer to achieve the highest possible homogeneity. Using the simplest technique possible, serum samples were diluted 1:30 (50 µl serum: 1450 µl buffer).

Due to the specific nature of the measurement, a tetramethylammonium hydroxide (TMAH) solution was used for dilutions. To improve the dispersion of dissolved blood components, a nonionic surfactant, Triton Χ-100, was added. This compound not only facilitates the dissolution of proteins, among other things, but also contributes to faster sample washout from the spectrometer's introduction system. An internal standard, rhodium (105Rh), was used to correct for matrix effects and instrument drift. Edetic acid (EDTA) was added to ensure the stability of metal ions dissolved in the solution. Furthermore, due to the presence of carbon-containing compounds, butanol was added to all solutions to compensate for the significant amount of carbon in the tested sample.

1.3 Measurement conditions

All determinations were performed using a quadrupole reaction cell spectrometer, the so-called DRC (Dynamic Reaction Cell) mode of the Elan DRC-e and NexlON 350D (PerkinElmer) apparatus with oxygen as the reaction gas.

PL 248990 Β1

1.4 Measurement validation

The following reference material, ClinCheck (Recipe, Germany), was used to validate the measurements. This is a reference standard commonly used in spectrometry, allowing for confirmation of measurement precision, sensitivity, and specificity.

Statistics

Differences in rates between the analyzed groups were assessed by multivariate analysis. The Kaplan-Meier curve was used for survival analysis.

Results

Analysis of the obtained results showed a significant relationship between the risk of death in women diagnosed with breast cancer and serum zinc concentration.

Women with breast cancer and with serum zinc concentration above 935.83 pg/l showed a significantly more than 3-fold reduced risk of death (group V vs. I: OR=3.19, p<0.01; 95%CI: 1.37-7.45).

Table 2. Incidence of death depending on serum zinc concentration in women with breast cancer.

Group Zn range pg/l Living Deaths OR p.value And 525.21-751.3 56 23 3.19 <0.01 II 752.8-819.31 65 14 1.68 0.26 III 819.57-870.36 63 15 1.85 0.18 IV 870.47-935.67 68 11 1.26 0.63 V 935.83-11044.98 70 9 Ref. Ref.

Women with breast cancer and serum zinc concentration above 1000 pg/l have a significantly more than five-fold reduced risk of death (group IV vs. I: OR=5.68, p=0.0003; 95%CI: 2.1-15.42).

Table 3. Incidence of death depending on serum zinc concentration in women with breast cancer.

Zn range pg/l Living Deaths

The figure shows the relationship between serum Zn concentration and the risk of death in women with breast cancer (Kaplan-Meier curve).

Literature

1. Plum LM, et al.: The essential toxin: impact of zinc on human health, Int J Environ Res Public Health. 2010Apr; 7(4): 1342-65

2. Puzanowka-Tarasiewicz H, et al: Biological functions of selected elements. Zinc - a component and activator of enzymes.

3. Zaichick VY, et al.: Zinc in the human prostate gland: normal, hyperplastic and cancerous. Int Uroi Nephrol. 1997; 29(5):565-74.

4. Siddiqui MK, et al. Comparison of some losing elements concentration in blood, tumorfree breast and tumor tissues of women with benign and malignant breast lesions: an Indian study. Environ Int. 2006 Jul; 32(5):630-7.

5. Pasha Q, et al. Statistical analysis of lost metals in the plasma of cancer patients versus Controls. J Hazard Mater. 2008; May 30; 153(3): 1215-21.

6. el-Ahmady O, et al.: Serum copper, zinc, and iron in patients with malignant and benign pulmonary diseases. Nutrition. 1995; 11(5Suppl):498-501.

7. Kuo HW, et al.: Serum and tissue loss elements in patients with breast cancer in Taiwan. Biol Losing Elem Res. 2002 Oct; 89(1):1-11.

8. Zhou W, et al.: Dietary iron, zinc, and calcium and the risk of lung cancer. Epidemiology. 2005 Nov ; 16(6): 772-9.

9. Zhang X, et al.: A prospective study of intakes of zinc and heme iron and colorectal cancer risk in men and women. Cancer Causes Control. 2011 Dec; 22(12): 1627-37.

10. Leitzmann MF, et al.: Zinc supplement use and risk of prostate cancer. J Natl Cancer Inst. 2003 Jul 2; 95(13).

11. Ananda S Prasad, et al.: Lessons learned from experimental human model of zinc deficiency, J Immunol Res. 2020 Jan 9; 2020:9207279

12. Lu Liu, et al.: Leveraging methylation to identify the potential causal genes associated with survival in lung adenocarcinoma and lung squamous cell carcinoma, Oncol Lett. 2020 Jul; 20(1): 193-200

13. Lingtao Su, et all.: Detecting cancer survival related gene markers based on rectified factor network, Front Bioeng Biotechnol. 2020 Apr 23; 8:349

14. Zhang M., et all.: A risk score system based on DNA methylation levels and a nomogram survival model for lung squamous cell carcinoma, Int J Mol Med. 2020 Jul; 46(1):252-264

15. Ruidan Li, et alk: GLI1 expression in pancreatic ductal adenocarcinoma correlates the clinical significance and prognosis: A meta-analysis, Medicine (Baltimore). 2020 Jun 26; 99(26):e20950

Claims (4)

Patent claims 1. A method for determining the risk of death in women with breast cancer, characterized in that it comprises determining the concentration of zinc in a serum sample of the tested person, wherein the concentration indicates a 3-fold reduced risk of death compared to the subgroup with the lowest serum zinc concentration (<751 pg/l), in the case of serum zinc concentration values above 935 μg/l. 2. A method for determining the risk of death in women with breast cancer, characterized in that it comprises determining the serum zinc concentration of the examined person, wherein the concentration indicates a 5-fold reduced risk of death compared to the subgroup with the lowest serum zinc concentration (<700 pg/l), in the case of serum zinc concentration values above 1000 μg/l. 3. The method according to claim 1 and 2, characterized in that the sample of biological material is serum from venous blood. 4. The method according to claim 1, 2 and 3, characterized in that the concentration of Zn in the sample is determined by direct measurement of Zn in serum from venous blood.